title: H000_total_R2
description: Protocols: Cells were collected at different time points after transdifferentiation induction: 0, 72 and 144 hours. Cells were sorted by Fluorescence Analysis Cell Sorting (FACS) prior to DNA extraction. Human BLaER1 cells were grown in RPMI medium (Invitrogen) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco), 2 mM L-glutamine, and 100 U/ml penicillin G sodium (Gibco). Cells were maintained at 37ºC in a humid atmosphere containing 5% CO2. Transdifferentiation was induced by addition of 100 nM of beta-estradiol (Calbiochem) and 10 nM of IL-3 and CSF-1 (Peprotech). Genomic DNA was extracted from the FACS sorted cells with the GeneJET Genomic DNA purification kit (Thermo Scientific) following the instructions of the manufacturer. A first PCR step was done by Phusion polymerase (Thermo Fisher) using 500 ng of genomic DNA and staggered oligo mix with the presence of 6% DMSO, annealing temperature of 60ºC and a total of 20 cycles of amplification. We used staggered oligos to avoid the same bases being read for the constant region during Illumina sequencing and to minimize technical issues during base calling. Up to 6 PCR reactions were combined, the amplicons were gel-purified, and 2 ng were used as a template for a second PCR. The second PCR step was also done by Phusion polymerase but without the presence of DMSO. We used Illumina barcoded oligos an annealing temperature of 60ºC and a total of 8 cycles of amplification. Samples were purified with Agencourt Ampure beads (Beckman Coulter), quantified with a Qubit fluorometer (Thermo Scientific) and checked for quality in a Bioanalyzer (Agilent).
attributes.tag: ENA first public
attributes.value: 2021-10-28
attributes.tag: ENA last update
attributes.value: 2021-10-28
attributes.tag: External Id
attributes.value: SAMEA8694963
attributes.tag: INSDC center alias
attributes.value: CENTER FOR GENOMIC REGULATION
attributes.tag: INSDC center name
attributes.value: CENTER FOR GENOMIC REGULATION
attributes.tag: INSDC first public
attributes.value: 2021-10-28T05:44:29Z
attributes.tag: INSDC last update
attributes.value: 2021-10-28T05:44:29Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10445:H000_total_R2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: BLaER1
attributes.tag: cell type
attributes.value: precursor B cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: chronic lymphocytic leukemia
attributes.tag: organism part
attributes.value: bone marrow
attributes.tag: sample name
attributes.value: E-MTAB-10445:H000_total_R2
attributes.tag: sex
attributes.value: female